CRISPR-Guided Non-LTR Retrotransposons for Targeted Gene Insertion
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome-editing technologies lack affordability, ease of setup, scalability, and the ability to target multiple positions within the eukaryotic genome effectively, limiting their application in genome engineering and biotechnology.
Innovation Solution
Employ engineered non-LTR retrotransposons fused with programmable DNA-binding proteins, such as CRISPR-Cas systems, to facilitate targeted transposition of donor polynucleotides into specific genomic locations, utilizing modified or truncated retrotransposon polypeptides and donor constructs for precise genome modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current genome-editing technologies are used, then targeted genome perturbations can be achieved, but they lack affordability, ease of setup, scalability, and ability to target multiple positions effectively
Solution Approach 1:
The patent combines CRISPR-Cas9 genome-editing technology with non-LTR retrotransposon transposition machinery into a unified system. The Cas9 protein and retrotransposon polypeptide are fused or functionally coupled, allowing the system to simultaneously perform targeted DNA cleavage and donor polynucleotide integration at multiple genomic loci through multi-component guide RNA structures
Solution Approach 2:
The engineered system serves multiple functions: it can target multiple positions in the genome simultaneously, perform both cleavage and integration functions, accommodate different donor polynucleotide types, and work with various guide RNA configurations. This multi-functionality addresses the limitation of conventional technologies that struggle with multi-position targeting
2Ease of manufacture
If current genome-editing technologies are used, then targeted genome perturbations can be achieved, but they lack affordability and ease of setup
Solution Approach 1:
The system divides the genome-editing function into modular components: separate Cas9 and retrotransposon polypeptide domains, distinct guide RNA regions for targeting and donor recruitment, and separable donor polynucleotide constructs. This modularity simplifies setup and allows flexible assembly while maintaining functionality
Solution Approach 2:
Guide RNA structures serve as intermediaries that bridge the Cas9 cleavage function and retrotransposon integration function. The multi-component guide RNAs mediate between the programmable nuclease and the transposition machinery, coordinating their actions at target sites and simplifying the overall system setup
3Productivity
If current genome-editing technologies are used, then targeted genome perturbations can be achieved, but they lack scalability
Solution Approach 1:
The system performs preliminary actions by pre-assembling multi-component guide RNAs that contain both targeting information and donor polynucleotide recruitment signals. Donor polynucleotides are prepared with complementary sequences beforehand, enabling rapid and scalable simultaneous editing at multiple positions without increasing operational complexity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient, targeted integration of long polynucleotide sequences into genomes, facilitating gene therapies and therapies like gain-of-function mutation correction and therapeutic transgene provision, with improved precision and scalability.
Implementation Method 1
a programmable DNA-binding protein configured to bind a target sequence within a target polynucleotide
Implementation Method 2
the non-LTR retrotransposon polypeptide facilitates targeted transposition of the donor polynucleotide into the target polynucleotide at the target sequence
Implementation Method 3
a CRISPR-Cas system comprising a Cas protein and one or more guide molecules capable of forming a complex with the Cas protein and directing sequence-specific binding of the complex to the target sequence
Data Source
AI summary
Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems and non-LTR retrotransposon elements.